The activity of replication origins is a major way of regulating DNA replication and the cell cycle. This process has been most well studied in the budding yeast Saccharomyces cerevisiae, where origins can be studied as autonomously replicating sequences (ARSs) on plasmids. However, DNA replication initiation mechanisms in other organisms are not well understeood. My preliminary data as well as published work from other labs suggests that replication initiation mechanisms differ among different budding yeast species. The recent abundance of computational tools and sequenced genomes allows the study of replication origins and their evolution across multiple species. I propose to isolate functional ARS sequences from different yeast species and, using comparative genomics, study the molecular determinants of ARS function. This will provide insight into both the mechanisms of ARS function as well as the evolution of replication initiation mechanisms. Using the well developed ARS assay combined with microarray analysis, I will isolate and map ARSs from different species of budding yeast. I will use comparative genomic approaches to characterize the mechanisms of ARS recognition utilized by the different yeasts. I will also assay the evolution of ARS elements and their genomic locations. To identify the differences in ARS recognition proteins that are responsible for differences in ARS function, I will perform a series of inter-species ARS and protein swapping experiments. Relevance: The initiation of DNA replication is a central player in genome dynamics and cell cycle regulation. It is therefore not surprising that defects in this process have been linked to genomic instability and cancer. The proposed studies will contribute to our understanding ofthe molecular mechanisms of replication initiation by tracking the co-evolution of ARSs and ARS recognition proteins across 200 million years of evolutionary time. In addition, we will learn how proteins from different yeasts interact with different ARS sequences. In the long-term, this information will be useful in understanding how DNA replication initiates in human cells and how defects in this process can contribute to cancer.